Immunobioinformatic approaches for development of multi-epitope vaccine against mpox targeting attachment, entry-fusion and immune evasion proteins of mpox virus

• A multi-epitope MXC25 vaccine (∼ 91.5 kDa) was constructed in-silico against MPXV • MXC25 projected 99.95% HLA-I and HLA-II combined global population coverage • K d 9.3×10 -13 M and docking energy -1518.1 kcalmol -1 were predicted for MXC25-TLR4 • MM-PBSA predicted binding free energy −124.29 kcalmol -1 for MXC25-TLR4 complex • Simulation of MXC25 injection at 1, 14, 56 days projected potential immune-response Recent outbreaks of the mpox virus caused a significant global health emergency with no specific vaccine available. Therefore, we employed immunobioinformatic approaches to design a multi-epitope vaccine against mpox targeting viral attachment, entry-fusion and immune-evasion proteins. The T-cell and B-cell epitopes were selected based on antigenicity, allergenicity, and IFN-γ inducing capacity to construct ∼91.5 kDa novel vaccine candidate designated as MXC25. The MXC25 showed non-toxicity, non-allergenicity, antigenicity 0.9007, pI 9.35, GRAVY -0.228, aliphatic index 76.13, and instability index 33.26. The three-dimensional structure of vaccine constructed using I-TASSER showed Ramachandran favored value 85.6%, outliers 4.03%, and Z-score −4.69. ClusPro-based molecular-docking of mpox recognition proteins (Toll-like receptor) TLR4-MXC25 complex displayed lower binding energy (-1518.1 kcalmol -1 ) and HawkDock-based higher affinity (K d 9.3×10 -13 M) relative to TLR2-MXC25 complex with -1322.1 kcalmol -1 binding energy and K d 9.7×10 -11 M. Molecular dynamic simulation further predicted binding affinity of TLR4-MXC25 complex. MM-PBSA estimated thermodynamic basis for binding free energy of −124.29 kcalmol -1 . Notably, MXC25 projected 85.96% HLA-I, 99.62% HLA-II, 99.95% HLA-I and HLA-II combined global population-coverage. Additionally, in-silico immune-simulations using three doses of MXC25 injected at 1, 14, and 56 days predicted favorable immune response plausibly for two years. Overall, MXC25 was predicted to have potential antigenicity, non-allergenicity, non-toxicity, high population coverage, immune response, in addition projected to confer structural and energetic stability upon binding with TLR proteins. Thus, the current computer-based simulations forecast properties of a probable antiviral against mpox for further extensive experimental validation and practical application towards combating reemerging global threat of mpox infection.